Microbial Detection and Quantification

ABSTRACT

Suitable dyes are used herein to indicate the presence of microbial contamination by spraying them onto surfaces in the form of an aqueous solution. The dye solution may also be allowed to dry, thus producing the dried residue of an aqueous solution. It&#39;s believed that these dyes change color in response to a change in polarity of the environment. Since water is a polar solvent and most bacteria are made from non-polar substances, the presence of bacteria changes the polarity of the environment, triggering a change visible by the unaided eye.

This is a Continuation-In-Part of co-assigned U.S. patent applicationSer. No. 10/737,674, filed Dec. 16, 2003 and claims the benefit offiling thereof.

BACKGROUND OF THE INVENTION

The invention concerns processes and products for the detection ofmicrobes like bacteria, yeast, mold and viruses.

In our everyday life we are unknowingly exposed tomicrobial-contaminated surfaces which can lead to illness. Studies haveshown specific bacteria-contaminated “hot spots” to include publictelephone, door handles, toys in doctors waiting rooms and child carefacilities, hot air dryers to dry hands, towels end sponges used in thekitchen, the hands of hospital staff during routine patient care andcross contamination from food preparation surfaces and knives where rawmeats and vegetables are mixed.

Recent bacterial contamination outbreaks in various locations in theUnited States alone have resulted in the death of children and seniorcitizens and sickening of others. Microbial contamination of food isalso a major problem throughout the world. Salmonella, E. coli and otherfood-borne bacteria cause untold numbers of illness each year. Acutesymptoms include nausea, vomiting, abdominal cramps, diarrhea, fever andheadache. Chronic consequences may follow after the onset of acutesymptoms. As cross-contamination of surfaces can cause transfer ofbacteria from meat, fish, and poultry to uncooked food such asvegetables, the ability to easily detect the presence of bacteria onfood-preparation surfaces would be of great benefit.

Similarly, the detection of harmful levels of microbes in the foodprocessing business is very important in maintaining the health offamilies and customers alike. In the food processing industry, bacteriamonitoring is critical. The processing of virtually all foods, from meatpacking to cheese production, involves monitoring microbes levels inorder to ensure the safety of the food supply.

The havoc wreaked by microbial contamination is not limited to the foodindustry alone. Recent decades have seen a dramatic rise in “superbugs,”a problem whose epicenter exists in the hospital and healthcarecommunity. The overuse of antibiotics as well as inadequacies inhospital cleaning have given rise to methicillin-resistant S. aureus(MRSA) and Clostridium difficile, as well as vancomycin-resistantenterococci and other gram-negative bacilli (Dancer, 2004). A recent BBCreport cited that MRSA claims an estimated 5000 lives yearly. Thearticle goes on to declare that “Cleanliness remains a major patientconcern and MRSA is a growing problem.” When one considers that manypatients in hospitals are already immuno-compromised and therefore atgreater risk of infection, the threat posed by nefarious bacteria in thehospital environment becomes even more menacing.

There are numerous reports and studies dedicated to the topic ofhospital cleanliness and the prevention of nosocomial infection.

Similarly, molds such as ergot have been known to grow in certaincereals such as rye and may be potentially hazardous by dint of theirproduction of toxic alkaloids similar to lysergic acid. Aspergillusniger and other molds have been known to produce spores that may causeallergic reactions as well as aggravate respiratory conditions such asasthma. A. niger may be particularly problematic if it begins to grow ona damp wall, or in air conditioning equipment in the home or commercialbuildings.

Certain yeasts, such as Candida albicans, can represent anothertroublesome class of microorganisms. C. ablicans has been associatedwith diaper rash in infants, oral thrush in children andimmuno-compromised adults, and vaginal yeast infections. Yeasts may alsoinfect the pharageal region of the body, as well as thegastro-intestinal tract.

Current methods of bacteria detection involve sampling the surfaces ofequipment. In a food processing environment, the equipment could be meatcutting machinery, whereas in a food preparing environment such as arestaurant or in the home, the surface could be a table, a cuttingboard, the inside of a refrigerator, or a work surface. The sample isthen incubated overnight to growth a culture. The overnight growthculture allows the sample to grow on an agar plate at appropriatetemperature and humidity so that the bacteria grow and multiply untilthey form colonies large enough to be visible to the naked eye. Afterincubating for the prescribed time and allowing the bacteria colonies togrow, the agar plate sample is examined manually and the colony formingunits (CFU) estimated by a trained technician. This method is somewhatexpensive and involves a substantial time lag; a time lag in whichcontaminated product may have been shipped or people exposed to microbespresent.

It is clear that there exists a need for a process and product whichallows for the rapid detection of harmful microorganisms.

SUMMARY OF THE INVENTION

In response to the foregoing difficulties encountered by those of skillin the art, we have developed an indicating composition that has amobile phase and a microbe sensitive colorant that undergoes a visiblydetectable change in the presence of microbes. The composition may beapplied to surfaces to reveal the presence of the microbes. The mobilephase may be a disinfectant. The colorant provides a color change thatis visible to the unaided eye in the presence of microbes. The mobilephase may be a liquid or gel and the colorant may be a dye. In someembodiments, the colorant changes color at a rate proportional to theconcentration of microbes. In other embodiments, the amount of microbepresent is proportional to a quantity of colorant that undergoes achange.

Examples of suitable dyes include merocyanine dyes,4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, zwitterionic dyes as exemplified by Reichart's dye, and others aswell as mixtures thereof. Of particular suitability are dyes that arezwitterionic, wherein the zwitterion is contained within a contiguous πelectron system comprising the dye chromogen. An additional class ofdyes that appear to be especially useful for microbe indicators aremerocyanine dyes.

The dye may also be applied to a surface as solvent-based or water basedsolution and allowed to dry, leaving the dried residue of the applieddye solution. The dried residue will change color upon contact withmicrobes and so may be used on packaging like facial tissue boxes, onmedical paraphernalia such as gloves, and on other surfaces which may beprepared with the dye before the material is used, and which willsubsequently indicate microbial contamination. Surprisingly, theinventors found that when these dyes are applied to a surface andallowed to dry, both the solvent used to make the coating and the use ofadditives such as hydroxypropyl-beta-cyclodextrin and surfactants had asignificant impact on the microbe detecting ability of the coating.

Hydroxypropyl-beta-cyclodextrin has been found to be effective inenhancing the brightness of the colorant after it is has been coatedonto a paper towel or similar wipe material. While not wishing to bebound by theory, we believe that the color of the dyes is improved bythe addition of a cyclodextrin derivative by inhibiting thecrystallization of the dye. Other chemicals may be added to a wipe tohelp prevent false positive readings due to the presence of bleach,which has been found to interfere with the dye.

Lateral flow devices incorporating microbe indicating colorants are alsoincluded within the teachings of the invention. These devices have amembrane having detection and control zones, where the detection zonechanges color in response to the presence of bacteria and the controlzone remains the original dye color to indicate that the assay isfunctioning properly.

Also described herein is a method for the detection of microbes onsurfaces by applying a solution containing a microbe-sensitive colorantto a surface and observing a visually detectable change indicating thepresence of microbes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of the five basic bacterial cell shapes.

FIG. 2 is a drawing of bacterial cell arrangements.

FIG. 3 is a drawing of merocyanine dye synthesis.

FIG. 4 is a drawing of merocyanine dye synthesis.

FIG. 5 is a drawing of the methylation of picoline.

FIGS. 6 A-D are diagrams of the indication of microbial contaminationusing aged chicken.

FIGS. 7 A-G are diagrams of a side-by-side indication of microbialcontamination indication and cleaning.

FIGS. 8 A-D are diagrams of the indication of microbial contaminationusing different concentrations of bacteria.

FIGS. 9 A-E are diagrams of the indication and quantification ofmicrobial contamination using bacteria and a titration of indicatingdye.

FIGS. 10 A-C are diagrams of the indication of microbial contaminationon a computer keyboard.

FIGS. 11 A-D are diagrams of the indication of microbial contaminationwith and without surfactant in the solution.

FIGS. 12 A-F are diagrams of the indication of the speed of indicationof microbial contamination depending on the solvent.

FIGS. 13 A-C are diagrams of the indication of microbial contaminationwhere the colorant is dried onto a substrate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the detection of bacteria and othermicroorganisms and the use of the term “microbe” herein should beunderstood to include bacteria, fungi like yeasts and molds, andviruses.

There are thousands of different kinds of bacteria. Some differ onlyslightly and it takes a highly trained person to identify them. Thereare also groups which differ greatly in growth habits and appearance andare quite easily identified. Regardless of minor differences, mostbacteria can be classified according to the five basic cell shapesillustrated in FIG. 1. From left to right in FIG. 1 the shapes are roundor cocci, rod or bacilli, spiral or spirilli, comma or vibrios, andfilaments.

In addition to their different shapes, their cell arrangement variesfrom diplococci, streptococci, and staphylococci (left to right in FIG.2). For example, some cocci are always grouped in pairs (diplococci).Others are arranged in chains (streptococci). Still others are bunched(staphylococci). Diplococci are the known to cause pneumonia.Streptococci are often associated with “strep throat.” Staphylococci arefamiliar to many because of their role in “staph infections” and sometypes of food poisoning.

Bacteria also vary somewhat in size, but average about 1/25,000 inch(2.54 cm) per bacteria. In other words, 25,000 bacteria laid side byside would occupy only one linear inch. One cubic inch is big enough tohold nine trillion average-sized bacteria—about 3,000 bacteria for everyperson on earth.

While there is great discussion on the theoretical basis of thesubdivision of bacteria based on modern molecular biological concepts,for the working microbiologist a rapid means of subdivision is on thebasis of the Gram reaction (a staining method to classify bacteria) andmorphology.

The gram-positive bacteria retain crystal violet stain in the presenceof alcohol or acetone. They include the important genera: Actinomyces,Bacillus, Bifidobacterium, Cellulomonas, Clostridium, Corynebacteriumk,Micrococcus, Mycobacterium, Nocardia, Staphylococcus, Streptococcus andStreptomyces. Some of the Gram-positive bacteria notably those of thegenera Corynebacterium, Mycobacterium and Nocardia retain dyes even inthe presence of acid. These are known as Acid-Fast bacteria.

The gram-negative bacteria do not retain crystal violet stain in thepresence of alcohol or acetone. They include the important genera:Acetobacter, Agrobacterium, Alcaligenes, Bordetella, Brucella,Campylobacter, Caulobacter, Enterobacter, Erwinia, Escherichia,Helicobacterium, Legionella, Nesseria, Nitrobact, Pasteurella,Pseudomonas, Rhizobium, Rickettsia, Salmonella, Shigella, Thiobacilus,Veiellonealla, Vibrio, Xanthomonas and Yersinia.

Bacteria membranes generally are made of lipid bi-layers ofliposaccharides. There are differences between gram-negative andgram-positive bacteria cell membranes, i.e, cell walls. The cell wall ofgram-negative bacteria is a thinner structure with distinct layers.There is an outer layer which is more like a cytoplasmic membrane incomposition with the typical trilaminar structure.

The main component of the gram-negative cell wall is lipopolysaccharide.Additionally there is present phospholipid, protein, lipoprotein and asmall amount of peptidoglycan. The lipopolysaccharide consists of a coreregion to which are attached repeating units of polysaccharide moieties.A component of the cell wall of most gram-negative bacteria isassociated with endotoxic activity, with which are associated thepyrogenic effects of gram-negative infections. On the side-chains arecarried the bases for the somatic antigen specificity of theseorganisms. The chemical composition of these side chains both withrespect to components as well as arrangement of the different sugarsdetermines the nature of the somatic or O antigen determinants, whichare such important means of serologically classifying many gram-negativespecies. In many cases it has been shown that the reason for certainorganisms belonging to quite different species, giving strongserological cross-reactivity is due to their having chemically similarcarbohydrate moieties as part of their lipopolysaccharide side chains,which generally have about 30 repeating units.

Gram-positive bacteria are characterized by having as part of their cellwall structure peptidoglycan as well as polysaccharides and/or teichoicacids. The peptidoglycans which are sometimes also called murein areheteropolymers of glycan strands, which are cross-linked through shortpeptides.

The bases of the murein are chains of alternating residues ofN-acetylglucosamine and N-acetyl muramic acid which are Beta-1,4-linked.The muramic acid is a unique substance associated with bacterial cellwalls. These chains are cross-linked by short polypeptide chainsconsisting of both L- and D-amino acids. While in gram-negative bacteriathe peptidoglycan is simple in structure and comparatively uniformthroughout most genera, in gram-positive bacteria there is a very bigvariation in structure and composition. In general the peptidoglycan ismultilayered. There have also been recorded some minor variations incomposition in some groups. Thus, in Mycobacterium and Nocardia theN-acetyl moiety of the muramic acid is replaced by the oxidized formN-glycolyl. The amino acid composition of the both the cross-linking aswell the stem polypeptides can vary extensively with different groups.These differences form the basis for the taxonomy of these organisms.

Molds and yeasts are organisms belonging the fungi kingdom. Althoughmany molds, and fungi are helpful to humans, several are pathogenic andcan release harmful mycotoxins which may result in poisoning or death.Yeasts can also lead to infection, the most widely known probably beingyeast vaginitis.

Zygomycota is a class of fungi which includes black bread mold and othermolds exhibiting a symbiotic relationship with plants and animals. Thesemolds are capable of fusing and forming tough “zygospores.” Ascomycotais a class of fungi, which includes yeasts, powdery mildews, black andblue-green molds, and some species which cause diseases such as Dutchelm disease, apple scab, and ergot. The life cycle of these fungicombines both sexual and asexual reproduction and the hyphae aresubdivided into porous walls which allow for passage of the nuclei andcytoplasm. Deuteromycota is another class of fungi which includes amiscellaneous collection of fungi that don't easily fit into theaforementioned classes or the Basidiomycota class (which includes mostmushrooms, pore fungi, and puffball fungi). These deuteromycetes includethe species that create cheese and penicillin, but also includesdisease-causing members such as those that lead to athlete's foot andringworm.

The use of dyes in the biomedical area has seen a remarkable growth inresearch interest and technical importance in the recent years. Dyes areused, for example, in many areas of analytical biochemistry, medicaldiagnostics and even in the treatment and prevention of disease. Thecolor of the dye is essential for certain applications and range fromsimple organic reactions for spectroscopic detection (U.S. Pat. No.5,036,000) and measurement of body fluid analytes (European Patent No. 0250 700) to high definition imaging technology for tumor detection(Motohashi, Med. Res. Rev., 11, 239, 1991). Dyes can also be usedclinically for the treatment of disease (U.S. Pat. No. 5,468,469).Photodynamic therapy (Sedlacek, “The change in research for the therapyof tumors”, Chimia, 45, 52, 1991) is successfully used in the treatmentof certain kinds of cancer; such as malignancies of the skin, head,neck, lung and esophagus. Other therapeutic applications are associatedwith the antiviral and bactericidal properties of dyes. Dyes are alsothe key agent in the important areas of histology, fluorescentbiolabeling and fluorescent bioprobes. The techniques involved arehighly sophisticated and require staining, washing and cross staining(Blum, Photodynamic action and disease caused by light” Reinhold, NewYork, 3, 1941).

The inventors have found that a microbe-indicating spray as well as arapid method for microbe quantification may be made using particularcolorants. Potential applications of this technology include but are notlimited to detection of microbes on solid surfaces such as counter-tops,hands, medical areas, bathrooms, bedrails, medical equipment, surgerytables, utensils, kitchens, food, food preparation surfaces, foodprocessing equipment, door knobs, phones and computer key-boards. Thiscolored dye coating, spray, or solution is sensitive to harmful levelsof bacteria and other microorganisms and the color change serves as avisual indicator tool to verify if cleaning and/or decontamination ofthe surface was effective.

The requirements for an indicating technology are fairly rigorous as thedye used must be sensitive to both gram-positive and gram-negativebacteria strains. The dye should rapidly interact with the microbe or amicrobial metabolite. For maximum versatility, the dye should also besensitive to other microorganisms, such as yeast and mold.

As mentioned earlier, dyes have been used for some time as stains forboth cell and bacteria identification. The stain solution reacts or ispreferentially retained by the cell or bacteria to help identify byimproving the contrast between it and the background or other componentspresent (Johnson, 1995). Usually, a stain has to be applied to asurface, then the excess removed by either shaking, or rinsing in orderto highlight the presence of microbes. The inventors are unaware of anyprevious reports of a colorant that changes color upon exposure to orupon interaction with microbes.

Solvatochromism may be responsible for the color changes seen, howeverthe inventors do not wish to be bound by one particular theory.Solvatochromic dyes undergo a color change when the molecularenvironment, such as solvent polarity and/or hydrogen bondingpropensity, changes. A dye may be blue in color in a polar environmentsuch as water, for example, but may be yellow or red in a non-polarenvironment such as a lipid-rich solution. The colors produced by such“suitable dyes” depend on the molecular polarity difference between theground and excited state of the dye as discussed more fully below.Reichardt's dye was selected as a model dye for investigation.

The inventors wondered if certain solvatochromic dyes might be useful todetect microbes by responding to the differences in polarity betweencertain cell components (such as the cell membrane, the cytoplasm, etc.)and the polarity outside of the cell. The inventors found that whenmicrobes were contacted with certain of these dyes coated ontosubstrates such as paper towels, a color change was indeed observed—notonly was there a color change, but in most cases the dye was deodorizedin the region contacted by the bacteria. To the surprise of theinventors, further research suggested that the mechanism may not beentirely attributed to solvatochromism. In fact, the inventors reporthere that, to their surprise, they found the following:

i) The quantity of the dye decolorized by bacteria or other microbes maybe correlated to the concentration of microorganisms exposed to the dye,suggesting that the method was quantitative vs. qualitative, and

ii) a range of microorganisms could be detected including gram positiveand gram negative bacteria, yeast, and mold, and

iii) the dyes tested could be used as a dry film coating or as asolution added to a liquid containing bacteria, or as a spray-ondetector system, and,

iv) when used as a dry coating on, for instance, a paper towel or anenamel surface, the properties of the solvent from which such dyes wereapplied significantly impacted the performance (decolorizing time,contrast between decolorized and non-decolorized areas, and sensitivity)of the detecting dye, and,

v) when used as a dry coating on, for instance, a paper towel, additivesincluded in the coating with the dye may impact the performance(decolorizing time, contrast between decolorized and non-decolorizedareas, and sensitivity) of the detecting dye. For instance,hydroxypropyl-beta-cyclodextrin enhances the performance of thedetecting dye, and,

vi) the bacteria induced decolorization of these dyes may be reversedusing a strong base.

While solvatochromism could contribute to the color changes observed,these observations may also be consistent with other plausiblemechanisms. For instance, these observations may also be consistent withan acid-base interaction of some type, or a proton donation reaction ofsome type that may contribute to color changes in the dye caused by thepresence of bacteria. The inventors have also not entirely ruled out thepossibility that a redox type reaction may also be contributing to theperceived changes in color when certain dyes are exposed to a range ofmicroorganisms. Other factors could also contribute to the color changesobserved with certain dyes in the presence of microbe, for instance,there may be an interaction with a portion of the cell membranes withcertain dyes that leads to color changes. Yet another possibility isthat the highly organized acid moieties on the bacteria cell walls maybe able to protonate certain indicator dyes, resulting in a loss ofcolor.

The inventors have discovered a surprising and as yet unexplainedphenomenon and used it to develop a method useful for the detection andquantification of a variety of microorganisms.

Generally, regarding visual detection of color changes; “color” is atype of sensation that arises when the machinery of the human eyedetects the presence or absence of light of various wavelengthsreflected or emitted from objects in the visual field. Light enteringthe eye is subjected to a spectral analysis by three types of retinalcone cells that are sensitive to specific regions of the visiblespectrum. Stimuli from these cells are in turn processed by retinalneurons, optic nerve neurons and the visual cortex such that a sensationof color is experienced. While several mechanisms exist to impart color(for instance, absorption, emission, fluorescence, phosphorescence,refraction, diffraction, etc.) the suitable focus is limited toabsorptive color. In other words, this invention relates to dyes thatowe their color to absorbing certain wavelengths of light.

Because of the way in which the human eye functions, the color perceivedis usually the complement of the color associated with the wavelength oflight being absorbed by the object. An object that appears to be red incolor when viewed in white light, for example, is in fact selectivelyabsorbing bluish light in the range of 490 to 500 nm wavelength.Similarly, an object that appears yellow in white light is in factabsorbing blue light in the range of 435 to 480 nm.

Absorption of visible light by molecules is associated with electronictransitions within the molecule and results in the generation of anexcited state. The energy difference between the ground state of themolecule and the relevant excited state determines the wavelength of thelight absorbed according to the Planck relationship:

E=hν

Where E=energy, h=Planck's constant, ν is the frequency of the photon oflight absorbed, and is related to wavelength λ and the speed of light cby:

ν=c/λ

A state diagram may be used to depict electronic transitionsgraphically:

Clearly, the energy of the photon absorbed is inversely proportional tothe wavelength of the photon. Thus, photons of blue light (435-480 nm)have higher energy than yellow light (580-595 nm). The color of a dye insolution or on an object when viewed under white light, therefore, isdetermined by the transition energy between the ground state of the dyemolecule and the first allowed excited state.

The light absorbing portion of a dye is conventionally known as thechromogen of the dye. The chromogen comprises a chromophore connected toa conjugated system. The chromophore is the group principally givingrise to the color of a dye, for instance, an azo group as in the case ofazo dyes, a polyene group, as in the case of carotene, carbonyl groups,as in anthraquinone and merocyanine dyes. There are many otherchromophores. Auxochromes influence the color and intensity of a dye byacting upon the conjugated chromogen. Auxochromes may or may not beconjugated with the chromogen. For instance, an amino group conjugatedto an azo group (chromophore) via, for instance, a benzene ring, willform an aminoazo chromogen. The conjugated amino auxochrome shifts theabsorption band of the azo group to longer wavelengths and increases theintensity of the absorption band. However, judicious placement of asulfonic acid group to an amino azo chromogen is not conjugated,however, the electron withdrawing effect causes a shift of absorption tolonger wavelengths.

An example of a dye that has a ground state more polar than the excitedstate is the merocyanine dye 1 as shown below. The charge-separated lefthand canonical 1 is a major contributor to the ground state whereas theright hand canonical 1′ is a major contributor to the first excitedstate.

Indigo 2, as shown below, is an example of a dye that has a ground statethat is significantly less polar than the excited state. The left handcanonical form 2 is a major contributor to the ground state of the dye,whereas the right hand canonical 2′ is a major contributor to theexcited state.

Suitable dyes for the practice of this inventions include thosediscussed above as well as Reichardt's dye, merocyanine dyes,zwitterionic dyes in which the formal positive and negative charges arecontained within a contiguous π-electron system,4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, and mixtures thereof. Merocyanine dyes fall within thedonor—simple acceptor chromogen classification of Griffiths as discussedin “Colour and Constitution of Organic Molecules” Academic Press(London) 1976, wherein a carbonyl group acts as an electron acceptormoiety. The electron acceptor is conjugated to an electron donatinggroup, for instance, an hydroxyl or an amino group that is able todonate electrons. Merocyanine dyes are a relatively broad class of dyesthat includes structure 3, wherein a nitrogen atom contained in aheterocyclic system serves as a donor, n may take any integer valueincluding 0. Merocyanine dyes have a charge separated (zwitterionic)resonance form.

Acyclic merocyanine dyes are also known, including vinylalogous amides.Merocyanine dyes have been studied for their ability to photosensitizesilver halide to certain wavelengths of light for use in photographicfilm. Many merocyanine dye structures are known. Structures 4-14 showseveral non-limiting examples of merocyanine dyes. Note that for each ofthese dyes, a charge separated resonance structure may be drawn. Theliterature suggests that the charge separated (zwitterionic) formcontributes significantly to the ground state of the dye.

where R may be methyl, alkyl, aryl, phenyl

Zwitterionic Chromogens

Certain dyes may be prepared that are permanently of a zwitterionicform. That is to say, these dyes have permanent charges associated withthe π-electron system and a neutral resonance structure for thechromogen cannot be drawn. Such dyes include Reichardt's dye 15, whichconforms to the general structure 16.

Additional non-limiting structures 17-25 may include the following,which conform to the general structure 16:

where X may be oxygen, carbon, nitrogen, sulfur.

The amount of dye must be sufficient to allow a change in color uponcontact with microbes where the change is detectible to the unaided eye,and so will depend on the sensitivity of the dye. The amount of dyefound to be sufficient is generally between 0.01 and 10 weight percent,more desirably between 0.05 and 5 and still more desirably between 0.1and 3 weight percent on a dry basis. The color change occurs quiterapidly in a manner that is dependent upon the concentration and type ofmicroorganism.

The composition includes the microbe-sensitive colorant as describedabove, and a mobile phase. The term “mobile phase” includes liquids andgases that may be used as carriers for the colorant. Acetonitrile,isopropanol, and mixtures of xylenes have been found to be suitablecarriers, though any effective carrier may be used. The mobile phase mayfurther also be a disinfectant or bactericidal composition.

The colorant dye may be in the form of a liquid that may be sprayed orwiped onto a surface to indicate the presence of microbes. Liquidcontaining the dye may be applied to surfaces and the applied liquidallowed to dry, forming the dried residue of the dye that may at a latertime be exposed to contamination by microbes. Upon exposure to microbes,the dried residue will change color, indicating the presence ofmicrobes. Such a method of indication using the dried residue of asolution applied dye may be useful for use on solid surfaces like, forexample, packaging such as facial tissue boxes, stickers, paper,tissues, medical paraphernalia like surgical gloves, surgical gowns anddrapes, face masks, head coverings like bouffant caps, surgical caps andhoods, examination and surgical gloves, footwear like shoe coverings,boot covers and slippers, wound dressings, bandages, sterilizationwraps, wipers, garments like lab coats, coveralls, aprons and jackets,patient bedding, stretcher and bassinet sheets, food preparation wraps,dish sponges, cloths, door handles, telephones, computer keyboards,computer mice, pens, pencils, notepads, toilet handles, wound dressings,bandages, and toys (e.g. in doctors waiting rooms, daycare facilities).

Substrates onto which the solvatochromic dye may be coated may thereforeinclude wipes, as well as other articles that may be exposed to bacterialike those mentioned above. The solvatochromic dyes may also beincorporated into lotions or cream used to check the hands for microbialcontamination. The dye may be incorporated into sponges or dish towelsto warn of contamination.

Substrates suitable for use as a wipe for coating with colorants includeany of those traditionally used for wipes including films, woven andnonwoven fabrics, cellulosic substrates like tissues, paper towels andcoform materials, airlaid materials, bonded-carded webs and so forth.Nonexclusive examples of substrates may be found in U.S. Pat. Nos.4,775,582 and 4,853,281, 4,833,003, and 4,511,438, all assigned to theKimberly-Clark Corporation.

A nonwoven fabric may be made according to processes like spunbonding,meltblowing, airlaying, bonding and carding, and so forth. Nonwovenfabrics may be made from thermoplastic resins including, but not limitedto polyesters, nylons, and polyolefins. Olefins include ethylene,propylene, butylenes, isoprene and so forth, as well as combinationsthereof.

“Spunbonded fibers” are small diameter fibers which are formed byextruding molten thermoplastic material as filaments from a plurality offine, usually circular capillaries of a spinneret with the diameter ofthe extruded filaments then being rapidly reduced as by, for example, inU.S. Pat. No. 4,340,563 to Appel et al., and U.S. Pat. No. 3,692,618 toDorschner at al., U.S. Pat. No. 3,802,817 to Matsuki et al., U.S. Pat.Nos. 3,338,992 and 3,341,394 to Kinney, U.S. Pat. No. 3,502,763 toHartman, and U.S. Pat. No. 3,542,615 to Dobo et al. Spunbond fibers aregenerally not tacky when they are deposited onto a collecting surface.Spunbond fibers are generally continuous and have average diameters(from a sample of at least 10) larger than 7 microns, more desirably,between about 10 and 20 microns.

“Meltblown fibers” means fibers formed by extruding a moltenthermoplastic material through a plurality of fine, usually circular,die capillaries as molten threads or filaments into converging highvelocity, usually hot, gas (e.g. air) streams which attenuate thefilaments of molten thermoplastic material to reduce their diameter,which may be to microfiber diameter. Thereafter, the meltblown fibersare carried by the high velocity gas stream and are deposited on acollecting surface to form a web of randomly dispersed meltblown fibers.Such a process is disclosed, for example, in U.S. Pat. No. 3,849,241 toButin et al. Meltblown fibers are microfibers which may be continuous ordiscontinuous, are generally smaller than 10 microns in averagediameter, and are generally tacky when deposited onto a collectingsurface.

As used herein, the term “coform” means a process in which at least onemeltblown diehead is arranged near a chute through which other materialsare added to the web while it is forming. Such other materials may bepulp, superabsorbent particles, natural polymers (for example, rayon orcotton fibers or other cellulosic materials) and/or synthetic polymers(for example, polypropylene or polyester) fibers, for example, where thefibers may be of staple length. Coform processes are shown in commonlyassigned U.S. Pat. Nos. 4,818,464 to Lau and 4,100,324 to Anderson etal. Webs produced by the coform process are generally referred to ascoform materials.

A bonded carded web is made from staple fibers which are sent through acombing or carding unit, which breaks apart and aligns the staple fibersin the machine direction to form a generally machine direction-orientedfibrous nonwoven web. Once the web is formed, it then is bonded by oneor more of several methods such as powder bonding, pattern bonding,through air bonding and ultrasonic bonding.

In the airlaying process, bundles of small fibers having typical lengthsranging from about 3 to about 52 millimeters (mm) are separated andentrained in an air supply and then deposited onto a forming screen,usually with the assistance of a vacuum supply. The randomly depositedfibers then are bonded to one another. Examples of airlaid teachingsinclude the DanWeb process as described in U.S. Pat. No. 4,640,810 toLaursen et al. and assigned to Scan Web of North America Inc, the Kroyerprocess as described in U.S. Pat. No. 4,494,278 to Kroyer et al. andU.S. Pat. No. 5,527,171 to Soerensen assigned to Niro Separation a/s,the method of U.S. Pat. No. 4,375,448 to Appel et al assigned toKimberly-Clark Corporation, or other similar methods.

The inventors have discovered that bleaches used to clean surfaces like,for example, sodium hypochlorite solution, chlorine, and sodiumbisulfite could possibly negatively impact solvatochromic dyes and causea color change even though bacteria is not present. Another aspect ofthe invention, therefore, includes a bleach detector colorant in a wipealong with solvatochromic dyes. The indicator could be, for example,2,2′,5,5′-tetramethyl benzidine, which is normally colorless and turnsred when exposed to chlorine or sodium hypochlorite. The indicator couldalso be a combination of starch and iodine which turns black in thepresence of chlorine or hypochlorite. Yet another indicator, fuchsine,may be useful for detection of sulfites, such as sodium metabisulfite.Fuchsine is pink and changes to colorless when exposed to sulfites. Inthis way, areas of the wipe may be designated as sensitive to bacteriaand other areas as sensitive to bleaches and preservatives so thatsurfaces containing active bleach give color change combinations thatallow the user to distinguish bacteria contamination from bleach. Thebleach indicator could be printed in a pattern to spell the word“BLEACH”, hidden on the wipe so that if the wipe were passed throughbleach, the word BLEACH would become visible, along with any other colorchange that the bleach may cause to the solvatochromic dye. The amountof bleach indicator need only be an amount sufficient to cause a colorchange that may be detected by the unaided eye and is in the same rangeas the solvatochromic dye.

The inventors also believe that it is possible to include small swatchesof a) a solvatochromic dye that detects bacteria, b) achlorine/hypochlorite detector material, such as tetramethyl benzidine,c) an oxidizing agent detector such as a mixture of starch and potassiumiodide, d) a bisulfite indicator such as fuschine, e) a nitritedetecting reagent, as examples, onto an indicating strip. In this way, avariety of quality indicators could give a status or quality of, forexample, food.

In another aspect of the invention, a coating on the substrate may beused to inhibit the detecting dye(s) from crystallizing, therebyobtaining a coating that has greater sensitivity to microbes. Ideally, acoating that has single dye molecules on the surface would have greatersensitivity for microbes. Each dye molecule would be free to interactwith the microbial membrane. In contrast, small crystals of dye firsthave to dissolve and then penetrate the membrane. While not wishing tobe bound by theory, we believe that hydroxypropyl-beta-cyclodextrin,hydroxyethyl-beta-cyclodextrin, gama-cyclodextrin,hydroxypropyl-gama-cyclodextrin, hydroxyethyl-gama-cylodextrin(hereinafter collectively “cyclodextrin”), all available from Cerestarinternational of Hammond, Ind., USA, hinder the crystallization of thedye, allowing a more vivid dye color to occur on the substrate. Theamount of cyclodextrin has been found to be effective is between 0.001and 2 weight percent, desirably between 0.01 and 1 weight percent andstill more desirably between 0.025 and 0.5 weight percent,

Certain surfactants have also been found to assist dyes in detectingmicrobes. Surfactants include the SURFYNOL® range of surfactantsavailable from Air Products and Chemicals, Allentown, Pa. and TWEEN® 80,a polyoxyethylene available from Fischer Scientific, Pittsburgh, Pa.

Another method of utilizing the detecting dyes to detect bacteria is inthe form of a lateral flow detection device. Such devices 2, as shown inFIG. 1, have a deposition pad, membrane, and a wicking pad, (not visibleseparately) though in some embodiments the deposition pad may beoptional. The device 2 has a detection zone 4 and a control zone 6. Inuse, a liquid sample is deposited on the device 2 in a sample area 8 andflows through the device 2 toward the wicking pad 10, passing throughthe detection zone 4. Sovatochromic dye is deposited in the detectionzone 4, in a line in this case though other shapes could be used, and inthe control zone 6, also in a line. As the sample moves through thedetection zone 4, if microbes are present, the dye in the detection zone4 will change color. The dye in the control zone 6 will not change colorsince the bacteria will be captured in the detection zone 4. The controlzone 6 is used to indicate that the assay is running properly. The colorof the detection zone 4 can be compared to the color of the control zone6 to indicate the relative magnitude of the microbes present. More thanone solvatochromic dye line may be used since the sensitivity of dyes todifferent microbes differs. Some dyes, for example, are more sensitiveto gram positive bacteria and some are more sensitive to gram negativebacteria. In this way more than one type of bacteria or other microbesmay be detected.

The following examples help to illustrate various embodiments of theinvention.

EXAMPLE MATERIALS

All reagents and solvents were obtained from Aldrich Chemical CompanyInc. (Milwaukee, Wis.) unless otherwise noted and were used withoutfurther purification. The microorganisms used in the study were:

1. Gram negative (viable)

-   -   Escherichia coli (ATCC #8739).    -   Pseudomonas aeruginosa (ATCC #9027)    -   Salmonella choleraesuis    -   Gardnerella vaginalis

2. Gram positive (viable)

-   -   Staphylococcus aureus (ATCC #6538)    -   S. Xylosis    -   Lactobacillus acidophilus

3. Gram positive (dead)

-   -   Staphylococcus Aureus (ATCC #6538)    -   S. Xylosis.

4. Yeast (viable)

-   -   Candida Albicans

5. Mold (viable)

-   -   Aspergillus Niger

Reichardt's dye (2,6-diphenyl-4-(2,4,6-triphenylpyridinio)-phenolate)and 1-docosyl-4-(4-hydroxstyryl)-pyridinium bromide were purchased fromAldrich Chemical Company of Milwaukee, Wis. The other merocyanines usedin this study were synthesized in-house and are described in detailbelow.

Synthesis of Merocyanine Dyes

1-Decosyl-4-(4-hydroxystyryl)-pyridinium bromide was commerciallyavailable from Aldrich Chemical Co. (Milwaukee, Wis.) and used directly.

Additional examples of merocyanine dyes were synthesized in thelaboratory in a two-step reaction.

The dye synthetically prepared using the synthetic method are shown inFIG. 3.

Methyl iodide was added slowly to a stirred solution of δ-picoline in 50ml of isopropanol in an ice-bath as shown in FIG. 4. After addition wascomplete, the reaction was heated to reflux and reflux continued for 2hours. The solution was then chilled in an ice-bath and the precipitatefiltered, and washed with chilled alcohol, on a Buchner funnel. Thepowder was then dried in the fume-hood for 2 hours. Yield of crudeproduct was 18.6 grams. The crude product was not further purified andused directly for the next step.

N-methyl-δ-Picolone (9.4 g, 0.04 mole), and vanillin (6.1 g, 0.04 mole)were all dissolved into 50 ml of ethanol with stirring as shown in FIG.5. To this solution was added piperidine (3.4 g, 0.04 mole) and themixture refluxed for 16 hours. The reaction mixture was then chilled inan ice-bath and the product filtered off using a Buchner funnel andwashed with chilled ethanol.

The crude dye of structure 13 above, where R=methyl, was then stirred in250 ml of 0.2 Molar potassium hydroxide solution for 60 minutes to formthe zwitterion and then filtered off using a Buchner funnel. The dye wasthen crystallized from the minimum quantity of a 1:1 water/methanolmixture. The yield was 9.4 g (98%).

The other dyes were synthesized in a similar manner starting from therespective alkyl iodide. The following Table 1 shows the compounds andthe yields obtained for dye structure 13 for three different R groups.

TABLE 1 The alkyl derivatives synthesized and the yields obtained ALKYLGROUP YIELD (%) R = METHYL 98 R = HEXYL 92 R = DODECYL 87

Example 1 Reichardt's Dye in Acetonitrile Solvent Applied to aPreviously Contaminated Surface

In order to investigate the use of these dyes as a spray, a solution ofReichardt's dye, (160 mg in 10 mL of acetonitrile) was made. A spraybottle with an aerosol propellant was utilized to create a spraymechanism.

A raw chicken leg was aged at room temperature for several days toensure high bacterial levels. As shown in FIG. 6, the chicken leg wasplaced on the surface of a ceramic plate for several seconds (6A), thenremoved, after which the surface was blotted to remove any trace ofchicken juice (6B). Next, the Reichardt's dye solution was sprayed ontothe surface using the spray bottle (6C). The test was repeated with enadditional piece of chicken to ensure repeatability and gave a similarresult.

After spraying the indicator dye solution onto the surface, the entirearea that had contained the chicken was decolorized (that is, theindicator spray color changed from blue to very pale or colorless),creating an outline of the chicken (6D). The dye was rapidlyde-colorized in the exact spot corresponding to where the chicken hadbeen placed on the surface.

Example 2 Reichardt's Dye in Isopropanol Applied to a ContaminatedSurface

Isopropanol was investigated as a carrier which could have additionalbenefits due to its disinfection capability. Reichardt's dye wasdissolved in isopropanol (160 mg dye into 10 mL of isopropanol). Plasticdoorknobs were utilized as a “real world” surface on which to testbacterial contamination. Juice from aged chicken was used to mark thesurface of one of the knobs. The other door knob was left uncontaminatedas a control. Both knobs were wiped to remove traces of contamination.The isopropanol solution of the dye was sprayed on both surfaces. Thecontaminated area of the doorknob was easily observed by itsde-colorization of the Reichardt's dye from blue to colorless.

Example 3 Reichardt's Dye in Isopropanol Indicator Spray Testing forFalse Positives on Contaminated Surfaces

The Reichardt's dye indicator has been shown to have high sensitivity tomicrobes from chicken. In order to test the indicator for falsepositives, other components of chicken fluid, such as lipids andproteins, were used. Chicken broth was utilized for its non-bacterialnature and high probability of containing potential interferences.

Swanson® Chicken Broth (Campbell Soup Co., NJ, commercially availablefrom retail grocery stores) from a freshly opened can was pipetted ontoa ceramic surface and wiped dry with a SCOTT® paper towel. Juice fromaged chicken was also pipette onto the ceramic surface at a differentlocation and wiped dry as a known positive control. The Reichardt's dyeindicator (160 mg in 10 ml of isopropanol) was sprayed over the ceramicsurface and it was clear that only the side containing the aged chickenjuice (and thus bacteria) was de-colorized. From this experiment it canbe concluded that, in the case of chicken, It is indeed the bacteriathat is triggering the de-colorization response and not some secondarycomponents such as chicken fat or proteins.

Example 4 Reichardt's Dye Indicator Spray as a Cleaning Aid on aContaminated Surface

The use of Reichardt's dye in isopropanol spray as a cleaning aid wasalso tested. As shown in FIG. 7, aged chicken juice was appliedlongitudinally to both halves of a square-shaped ceramic surface (7A).On one half, side A, no cleaning was performed other than vigorouslywiping the surface with a SCOTT® paper towel. On the other half, side B,Kimberly-Clark Professional Moisturizing Instant Hand Antiseptic (60%ethanol solution, Roswell GA) was applied to the towel and used to deanthe surface (7B). The Reichardt's dye spray was then applied todetermine if cleaning had a difference (7C). It was clear that althoughthe cleaner had helped, several areas were “missed” (7D). Next, thebottom halves of both striped areas were cleaned vigorously with the K-CProfessional Antiseptic again, this time using the cues from the sprayto guide where cleaning was most necessary (7E). When the area wassprayed again (7F), no de-colorization took place, verifying thecleanliness of the surface (7G).

Example 5 De-Colorization of Reichardt's Dye-Coated Paper Materials

This experiment tested the ability of a surface coating of Reichardt'sdye to respond to bacterial contamination. As shown in FIG. 8, a sheetof paper was also brush-coated with a Reichardt's dye solution (80 mg/10mL acetonitrile) (8A). To this paper was added 100 μL aliquots of 10⁷,10⁶, 10⁵, and 10⁴ CFU/mL E. coli or S. aureus solutions (8B). Water wasused as a negative control. The dye color was rapidly discharged whencontaminated by both types of bacteria (8C), but more rapidly for the S.aureus (8D). It was later determined that while both bacteria solutionswere indeed at 10⁷ CFU/mL concentrations, the actual concentration ofthe S. aureus solution was 7×10⁷ CFU/mL compared to 1×10⁷ CFU/mL for theE. coli solution. Water caused slight de-colorization of the dye afterseveral minutes, in contrast to the rapid de-colorization (<1 min)observed for the bacteria solutions.

A sheet of paper self-adhesive stickers (Avery-Dennison) was alsobrush-coated with two different concentrations of Reichardt's dyesolution (160 mg/10 mL acetonitrile, 80 mg/10 mL acetonitrile). Thestickers were applied to the lid and latch mechanism of a Huggies® WetWipes box. A gloved hand was used to transfer 10⁷ CFU/mL S. aureus tothe surface of the stickers. Though both concentrations were rapidlyde-colorized, the color discharge was more easily visible on the surfacewhich had the lower concentration of dye, indicating that there may bean optimal coating concentration that provides detection and strongvisual contrast. The sticker could provide a means for easy and rapiddetection of bacterial contamination for a variety of applications.

Example 6 Quantification of Bacteria Concentration Using Reichardt's Dye

A new potential for the Reichardt's dye-based bacterial indicator wasrealized through testing of liquid dye with bacteria on a substraterather than substrate-bound dye coming into contact with liquid bacteria(Example 5). This experiment focused on determining how the dye solutionresponds to known concentrations of bacteria residing on a surface. 100μl of 10⁸ CFU/ml gram-positive bacteria was placed on a SCOTT® towel(FIG. 9A). To this spot was added a drop of the Reichardt's dyedissolved in acetonitrile (160 mg in 10 ml acetonitrile) (9B).

For comparison, a spot of dye was dried onto the towel and the sameamount of bacteria was added. Upon addition to the spot of bacteria,Reichardt's dye was immediately de-colorized. The reaction of bacteriaplaced onto the dye-containing cellulosic towel, by contrast, takesseveral minutes to de-colorize. Additional drops of the dye were addedto the spot of bacteria (9C), and de-colorization continued until thefourth drop, at which point the purple color persisted (9D). Attempts torestore the dye color on the dye-coated SCOTT® towel by addingacetonitrile using a pipette were unsuccessful (9E).

Example 7 Bacterial Indicator Titration Testing Using Reichardt's Dye

The discovery of rapid de-colorization of the dye solution bysubstrate-bound bacteria, as well as the fact that the reaction reachedan end point, prompted exploration into the ability of the dye to givequantitative information about bacterial CFU/ml. The purpose of thisexperiment was to titrate various concentrations of substrate-boundbacteria with dye and determine if the amount of dye required tostabilize the color varied at all with bacterial CFU/ml.

On a SCOTT® paper towel was placed 100 μl each of serially diluted S.aureus bacterial suspensions. Drops (10 μl) of the Reichardt's dye inacetonitrile solution (40 mg/10 ml) were then pipetted Onto each spotwhere the bacteria was placed. The dye solution was at first colored butwas almost instantly (<1 sec) decolorized and additional drops wereplaced on the same spot until the dye was no longer de-colorized and thepurple/blue color did not fade. This was repeated on each spotcorresponding to where different concentrations of bacteria were placed.

The results showed a good correlation to the level of bacterialcontamination on a surface or substrate.

Example 8 Bacteria Titration in Aged Female Urine

To illustrate the practical use of this new method, a sample (100 μl) ofaged, pooled female urine was placed on a cellulosic towel to yieldseveral spots each having 100 μl volumes of the urine. Two solutions ofthe dye were used for a titration study; 40 mg dye/10 ml acetonitrileand 160 mg dye/10 ml acetonitrile. The dye solutions wore then placedonto the urine spots in 10 μl aliquots and continued until theblue/purple dye color remained (that is, dye was added to the urineuntil the color persisted). Table 2 presents the volume of each dyesolution required for the dye color to remain steady (that is, no longerdepolarized). Aged female urine is known to have high bacterialcontamination and this preliminary study shows a high level ofcontamination in this particular sample.

TABLE 2 Bacterial Quantification of Female Pooled Urine SAMPLE 4 mg/mlDye Solution 16 mg/ml Dye Solution Urine 120 μL 30 μL

It is interesting to note that it took four times more dilute (four folddilution) dye solution than the more concentrated (four fold higher) dyesolution. This may allow tailoring of the indicator system for thevarying CFU levels seen in different industries (food vs. healthcare,etc) by using maximal dye concentrations to minimize the amount requiredfor saturation. For example, chicken parts may produce anywhere from10²-10⁹ bacteria levels, depending on time and storage conditions. Foodpreparers and handlers, however, might only be concerned about levels ofbacteria 10⁷ and higher out of concern for illness. Hospitals, on theother hand, are typically treating patients who may already beimmune-compromised in some way, either because of disease, illness, orsurgery, Hospital staff may therefore be concerned about much lowerlevels of bacteria than most other industries and could potentiallybenefit from an indicator dye concentration tailored to their specificneeds in order to reduce infection risk to susceptible patients.

Example 9 Testing Bacteria Indicator with a Variety of MicroorganismsIncluding Bacteria, Mold, and Yeast

In the same manner as previously described, a cellulosic towel was usedas a substrate onto which bacteria and other microorganisms werepipetted. 10⁷ CFU/ml of S. aureus, C. albicans (yeast), G. vaginalis, E.coli, P. aeruginosa, and L. acidophilus were pipetted onto the towel(100 μl each). In addition, 10⁵ of A. niger (a common mold) was alsopipetted onto the towel. Reichardt's dye solution (160 mg in 10 ml ofacetonitrile) was then added in 10 μl aliquots to each spot and thenumbers of drops needed to establish a persistent color were counted.

The amount of dye required to maintain a persistent purple color foreach organism is provided in Table 3. The strongest reaction wasobserved with L. acidophilus, followed by S. aureus, G. vaginalis, E.coli, P. aeruginosa, C. albicans, and finally A. niger. Although thereseemed to be as strong a reaction for the gram-positive S. aureus as forthe gram-negative G. vaginalis

TABLE 3 Titration of Various Microorganisms with Reichardt's Dye Amountof Dye Required for Persistent Compound Type Color (μl) LactobacillusGram(+) 110 S. aureus Gram(+) 90 G. vaginalis Gram(−) 90 E. coli Gram(−)80 P. aeruginosa Gram(−) 80 C. albicans yeast 70 A. niger Mold 50

Example 10 Testing of Reichardt's Dye Indicator with Bacterial Cell-WallComponents

Insight into how this indicator technology works was obtained byutilizing molecules commonly found in the cell walls of bacteria.Although there is some commonality in the compounds which comprise thesurfaces of gram-positive and gram-negative bacteria, their arrangementand chemical composition differ from one another. Gram-negative bacteriahave an outer membrane coated with lipopolysaccharide (LPS). LPS lends anet-negative charge to the surface of gram-negative bacteria andcontributes to its pathogenesis. Gram-positive bacteria are coated witha thick peptidoglycan, or murein, sheet-like layer. The sheets areformed from alternating N-acetylglucosamine and N-acetyl muramic acidmolecules. Teichoic acids are also found in gram-positive bacteria andmay be linked to the N-acetylmuramic acid. While gram-negative bacteriaalso have peptidoglycan, the layer on gram-positive bacteria is muchthicker. Furthermore, the peptidoglycan layer of gram-negative bacteriais located underneath the LPS layer, making it less accessible from thesurface.

Onto a SCOTT® paper towel were placed Solutions of E. coli-deriveddetoxified lipopolysaccharide (Lipid A component removed), lipoteichoicacid derived from Streptococcus faecalis, E. coli-derivedlipopolysaccharide, and muramic acid. With the exception of the pureLPS, all solutions were prepared in 5% (wt/wt), 1% (wt/wt), and 0.2%(wt/wt) concentrations. Pure LPS was prepared in 0.1% (wt/wt), 0.02%(wt/wt), and 0.004% (wt/wt) for safety reasons. Reichardt's dye (160 mgin 10 mL acetonitrile) was added in 10 μl aliquots to each spot andamount of dye required to produce a persistent color was recorded. Thereverse experiment was also conducted where the cell-wall compounds wereplaced onto a spot of dye on the paper towel.

Muramic acid produced the strongest reaction, resulting in a nearinstantaneous de-colonization of the dye in both experimental set-ups.The other compounds did cause eventual de-colonization of the dye, butdid not appear to react as strongly as muramic acid. Because muramicacid is found in greater concentrations on gram-positive bacteria, theseresults demonstrate the potential of this dye to not only give CFU/mLdata, but also the potential to distinguish between gram-positive andgram-negative bacteria based on strength and speed of reaction.

Example 11 Testing of Chicken-Related Components

The Reichardt's dye indicator has been shown to have high sensitivity tomicrobes growing on raw chicken meat that has been stored at roomtemperature. With consideration to the potential for false positives,however, it became necessary to test the response of the indicator toother components of chicken fluid, such as lipids and proteins. Cannedchicken broth was utilized as a control that would contain chickenderived products such as lipids, protein, etc., to check for potentialinterferences from these naturally occurring materials.

Freshly opened Swanson® Chicken Broth was pipette onto a hot platesurface and wiped dry with a SCOTT® towel. Juice from raw chicken thathad been stored at room temperature for several days was also pipettedonto the hot plate and wiped dry as a positive control. The Reichardt'sdye indicator (160 mg in 10 ml of isopropanol) was sprayed over thesurface and it was clear that only the side containing the aged chickenjuice (and thus bacteria) was de-colorized. From this experiment it canbe concluded that, in the case of chicken, it is indeed the presence ofmicrobes that is triggering the de-colorization response and not someother component such as chicken fat or proteins.

Example 12 Effect of Strong Base on Reichardt's Dye De-Colorization

Preliminary results in regard to the interaction of Reichardt's dye withcell wall components such as muramic acid, as well as work directed atidentifying potential false positives suggested that a reaction withacids may contribute towards de-colorization of Reichardt's dye. Thisled to speculation that an acid-base reaction may play a part in thecolor change observed. An experiment to test the effect of a strong baseon decolorized Reichardt's dye was planned.

Several drops of Reichardt's dye (160 mg in 10 ml of acetonitrile) werepipetted onto a SCOTT® towel and allowed to dry. Two compounds known tocause color changes (acetic acid and Aldrich buffer pH 2.0) were eachdropped onto two of the spots which led to rapid de-colorization of thedye. A drop of 1 N NaOH was then pipette onto one of each of the spots,causing rapid re-colorization. The blue/purple color of Reichardt's dyereturned after the 1 N NaOH was added.

A second experiment was performed using the indicator spray tocorroborate these results. Aged raw chicken juice was pipetted onto thehot plate surface in an easily recognizable pattern. The surface wasblotted dry and sprayed with Reichardt's dye indicator spray (160 mg in10 ml acetonitrile), causing de-colorization of the dye in the exactform of the pattern of chicken juice. A drop of 1 N NaOH was then placedon an area that was previously decolorized, leading to re-colorizationof that small spot. This was repeated with another area.

To test the possibility that the 1 N NaOH was simply acting on thebacteria and not the dye, aged chicken juice and 1 molar NaOH were mixedin equal proportions and allowed to stand for 30 seconds. This mixturewas then used to create another identical (though smaller) pattern. Thissolution also caused rapid de-colorization of Reichardt's dye, however,the color returned upon addition of 1 N NaOH.

Example 13 Testing of Reichardt's Dye-Coated Stickers with Normal andBacterial Vaginosis (BV)-Infected Vaginal Fluid

Considering the high prevalence of vaginal infections of bacterialorigin, an experiment was performed to determine the response ofReichardt's dye-coated stickers to healthy (low pH, no bacterialinfection), a pH positive/BV negative (no bacterial infection, buthigher than normal pH), and pH positive/By positive (higher than normalpH and known bacterial infection) vaginal fluid samples. A sheet ofstickers was brush-coated with two different concentrations ofReichardt's dye solution (160 mg/10 acetonitrile, 80 mg/10 mLacetonitrile, 40 mg/10 mL acetonitrile, 20 mg/10 mL acetonitrile). Asticker of each concentration was tested with normal, BV positive 1 pHpositive, and BV negative 1 pH positive vaginal fluid samples.

Normal vaginal fluid yielded the sharpest de-colorization of the dye,presumably due to the combination of lactobacillus and low pH. The BVpositive/pH positive sample exhibited the next sharpest de-colorization,perhaps due to the presence of Large numbers of BV bacteria. The BVnegative/pH positive sample only faintly de-colorized the dye, perhapsdue to a lesser amount of lactobacillus than in the normal sample. Thethree states of de-colorization were easily distinguishable, suggestingthat there may be a diagnostic potential for this technology within thevaginal health field.

Example 14 Testing of Common Surface with Reichardt's Dye IndicatorSpray

It is well known that bacteria can survive on dry surfaces for hours, ifnot days. The ability to identify bacteria and other microbes on commonsurfaces and alert consumers or workers to contamination would aid incleaning and disinfection and help minimize the spread of infection.

As shown in FIG. 10, an old computer keyboard was used a model“real-world” surface to test the microbial indicator spray (10A).Reichardt's dye was dissolved in isopropanol (160 mg in 10 mL ofisopropanol) and attached to an aerosol-based spray device. The keyboardwas then sprayed with the indicator solution (10B).

Spraying of the keyboard with the Reichardt's dye indicator solutioncaused rapid de-colorization of the dye in certain areas (10C). It isinteresting that only certain keys or areas showed contamination,allowing for specific identification of keys that are highly sullied,such as the number pad. As keyboards are used quite often, but rarelycleaned, this surface indeed provides a glimpse of microbial levels onreal-world surfaces.

Example 15 Use of a Surfactant to Enhance Sensitivity of the BacterialIndicator

A solution of Reichardt's dye (80 mg/10 mL acetonitrile) and TWEEN® 80(200 μL) polyoxyethylene surfactant (from Fischer Scientific,Pittsburgh, Pa.) was prepared. This solution was then used to coat aceramic surface (FIG. 11) and allowed to air dry. A second solution ofReichardt's dye (80 mg/10 mL acetonitrile) without surfactant was placedon the surface and allowed to air dry as well (11A). After drying, adrop of aged chicken Juice known to have a high bacterial count wasplaced on the each coating area (11B). The area containing the TWEEN® 80surfactant (11C) de-colorized at a much faster rate (>20-30 seconds)when compared to the area that did not contain the TWEEN® surfactant(11D). Furthermore, the addition of TWEEN® surfactant allowed for easyremoval of the dye from the surface. The addition of a small amount ofwater allowed for complete removal from the surface while the additionof water to the spot that did not contain the surfactant did not improveease of removal from the surface.

Example 16 Importance of Solvent Choice

The behavior of Reichardt's dye coatings made using various differentsolvents was evaluated.

Solutions of Reichardt's dye in acetonitrile, isopropanol, and xyleneswere prepared and the solutions were used to coat SCOTT® kitchen rollstowels and allowed to air-dry (FIGS. 12A and 12D). The treated towelshad 100 μl aliquots of S. aureus placed on them (12B,E) and the coatingwas observed for a color change. Only the acetonitrile solution-basedcoating had a rapid decolorization where the bacterial suspension wasplaced (12C), The Reichardt's dye was observed to have an even colorwhen dissolved in acetonitrile. No visible color change was observedwith the other two solvent coatings (12F).

The inventors found that the concentration of Reichardt's dye could beadjusted such that isopropanol could be utilized as a solvent for thedye. Though the color of the dye is less intense than that seen withacetonitrile, the decolorization in response to microbial contaminationis readily and easily observed.

Example 17 N-docosyl-merocyanine Dye Coated onto Cotton Fabric

The transparent film covering half of a fresh chicken on a polystyrenetray (from supermarket) was stored at roam temperature for three weeks.The pale yellow juices that collected in the polystyrene tray werecollected using a pipette and used for tests.

47 mg of 1-docosyl-4-(4-hydroxystyryl)-pyridinium bromide (from AldrichChemical) was mixed with 10 g dimethylformamide. A small amount ofsolids remained after shaking and allowing to settle. The orangesupernatant fluid was dropped onto woven cotton fabric of basis weight(29.2 cm×20.3 cm=6.888 g) to make orange-yellow colored circles. Onedrop of 1N sodium hydroxide solution was added to one orange-yellow spoton the cotton fabric, changing the color from orange-yellow to a pinkishorange.

Aged chicken juice was spotted onto the orange-yellow spots on thecotton fabric, producing a color change to very pale yellow. The colorchange was rapid on cotton. Similarly, aged chicken juice was droppedonto the pinkish-orange areas on the cotton (dye+NaOH soln.) causing asimilar color change from pinkish-orange to very pale yellow.

Example 18 N-methylmerocyanine Coated onto Kitchen Paper Towel & AgedUrine

Female human urine was collected and stored in a glass jar at roomtemperature for 8 days. N-methyl merocyanine dye of the followingstructure was synthesized as described above. 0.5 g was dissolved in 20ml deionized water and coated onto a SCOTT® kitchen roll paper towel bydipping the towel into the solution, allowing the excess to drip off,and then allowing the coated toweling to dry at ambient conditions. Thepaper towel was stained a deep orange color by the dye.

Aged urine was dropped onto the orange colored towel to give animmediate color change from deep orange to pale yellow. As a control,the aged urine was filtered through a 0.2 micron filter to removebacteria and other microbes. After filtration, the aged urine did notcause a color change when dropped onto the towel suggesting thatmicrobes were responsible for causing the color change vs. othercomponents in the aged urine.

Example 19 N-methylmerocyanine Coated onto Kitchen Toweling & Aged Urine

Female human urine was collected and stored for 24 hours at 37° C.Pooled female urine may be expected to have a bacterial loading ofapproximately 1×10⁵ CFU/ml after storage under these conditions.N-methyl merocyanine dye of the following structure 26 was synthesizedas described above. 0.5 g was dissolved in 20 ml deionized water andcoated onto a SCOTT® kitchen roll paper towel by dipping the towel intothe solution, allowing the excess to drip off, and then allowing thecoated toweling to dry at ambient conditions. The paper towel wasstained a deep orange color by the dye.

The aged urine was dropped onto the orange colored towel to give animmediate color change from deep orange to pale yellow. As a control,the aged urine was filtered through a 0.2 micron filter to removebacteria and other microbes. After filtration, the aged urine did notcause a color change when dropped onto the towel suggesting thatmicrobes were responsible for causing the color change vs. othercomponents in the aged urine.

Example 20 N-methyl Merocyanine Dye Coated onto Kitchen Paper Towels andPet Bird Bowel Movements

Budgerigar feces was collected from a caged pet budgie, and shaken inapproximately 10 ml of Atlanta city domestic tap water. N-methylmerocyanine dye of structure 26 above was synthesized as describedabove. 0.5 g was dissolved in 20 ml deionized water and coated onto aSCOTT® kitchen roll paper towel by dipping the towel into the solution,allowing the excess to drip off, and then allowing the coated towelingto dry at ambient conditions. The paper towel was stained a deep orangecolor by the dye (FIG. 13A).

Drops of the budgie feces suspension in tap water were spotted onto thecoated towel (13B) and produced an immediate color change from deeporange to pale yellow where the suspension was added (13 C). As acontrol, Atlanta city domestic tap water was dropped onto a differentarea of the towel and while the color was diluted somewhat by the water,the area remained orange.

Example 21 (Prophetic) Indicator Dyes Used in a Coating

1 gram of hydroxypropyl methyl cellulose, 0.5 gram N-methyl merocyanineof structure 26 may be dissolved in a mixture of 10 grams of water and10 grams of isopropyl alcohol with good stirring. This solution may becoated onto polyester film and allowed to dry at room temperature toproduce a coated flexible film capable of detecting the presence ofmicrobes.

Example 22 (Prophetic) Indicator Dyes in a Coating

1 g of ethylcellulose, 0.25 g, N-methyl merocyanine of structure 26 maybe dissolved in 20 grams of tetrahydrofuran. This solution may be coatedonto polyester film and allowed to dry at room temperature to produce acoated flexible film capable of detecting the presence of microbes.

Example 23 Lateral Flow Device

Millipore nitrocellulose HF75 membrane (from Millipore Corporation ofBillerica, Mass., USA) was laminated onto a plastic supporting card(from Millipore Corp.) having a length of approximately 30 centimeters.On both the detection zone and control zone, a solution of 5 weightpercent Reichardt's dye in isopropanol was hand striped. The membranewas dried in a laboratory oven for 1 hour at a temperature of 37.5° C.After the membrane card was taken from the oven, a cellulosic wickingpad (Cat# CFSP203000 from Millipore Corp.) was attached to one end ofthe membrane closer to the control zone. The other end of the card, usedto attach the sample pad, was cut off. The card was then sliced into 4mm strips to form half sticks.

Once the half sticks were prepared, a bacteria solution was applied tothe end of the detection membrane. Capillary action pulled the solutionand bacteria into the detection zone and a color change was noted in thedetection zone. The control line color remained the same through out thetest.

Example 24 Cyclodextrin Enhancement

A SCOTT® paper towel was first coated withhydroxypropyl-beta-cyclodextrin (from Cerestar International, Hammond,Ind., USA) in solution in water (1 gram in 20 ml) by dipping andair-drying at ambient temperature. When dry the coated paper towel wastreated with a solution of Reichardt's dye in isopropanol (1 weightpercent) and allowed to air-dry. The dried towel was a purple/blue incolor. Here the cyclodextrin hinders the crystallization of the dyeallowing a more vivid color of the dye to occur on the paper towel. Thiscoated towel was used in a test with gram-negative bacteria (E. Coli)and found to turn colorless in less than 5 seconds when an aliquot of100 microliters of media containing 10,000 CFU/ml was applied to thetowel. This decolorization was found to occur down to the bacteriaconcentration of 500 CFU/ml, though this took as long as 15 seconds.Thus by hindering the dye from crystallizing, the dye is believed to bepresent on the substrate as single molecules and therefore thesensitivity of the dye to bacteria levels increases. The inventorsbelieve that by careful use of a coating (e.g. cyclodextrin) on thetowel a mono-molecular coating of dye will occur on the surface of thesubstrate and maximum sensitivity will occur.

Example 25 Dry Sample Testing

A test using the Reichardt's dye coated paper towel with a “dry”bacteria sample, not in solution was carried out. A dry sample of acolony of E. Coli bacteria lifted off an agar petri dish containing aseries of growing cultures was used. This dry sample was then rubbedonto a pre-moistened dye coated SCOTT® paper towel. The area where thecolony was placed and rubbed turned colorless within 1-5 seconds. Thisis a similar to how a wet wipe towel would be used and it performedwell.

Example 26 Bleach Indicator Test

A mixture of Reichardt's Dye and 3,3′,5,5′-tetramethylbenzidine (TMB)was coated onto a SCOTT® paper towel and allowed to air-dry. A dilutebleach solution was applied to the paper towel which resulted in theReichardt's dye de-colorizing and the TMB turning orange/yellow color.This shows that a bleach indicator can be built into a bacteriaindicating wiper.

In the final test, a SCOTT® paper towel having a coating of Reichardt'sdye and TMB chemistries was exposed to suspension of E. Coli bacteriadrop-wise. The towel area that came in contact with the bacteriadecolorized to a white spot in less than 10 seconds. No orange/yellowcolor was observed to develop.

Example 27 UV-Vis Absorption Spectra of Merocyanine and ZwitterionicDyes

Reichardt's dye was used without further purification. N-n-hexyl andN-n-dodecyl merocyanine dyes were synthesized as described. Solventsused were obtained from Aldrich Chemical and were HPLC grade. A ShimadzuUV-1601 UV-Visible Spectrophotometer (Shimadzu Corporation) was used tomeasure the longest wavelength peak absorption of the dyes in the rangeof 400 to 800 nm, dissolved in three different solvents, contained inquartz curvettes. The following table contains the results of thetesting with the solvents on the left side and dyes across the top.

Dodecyl Hexyl Merocyanine merocyanine Reichardt's dye Acetone 617.5 nm(Green) 617 nm (Green) 674 nm (bluish green) Methanol 514 nm (Orange)522 nm (Orange) 509 nm (red) Acetonitrile 582 nm (Greenish- 600 nm(Blue) 623 nm (Blue) blue)

The merocyanine dyes also showed absorption near 400 nm, in addition tothe longer wavelength absorption, which altered the perceived color.

Clearly, based upon the spectroscopic measurements, these dyes showlarge shifts (>10 nm) in maximum wavelength peak absorption betweenthese microbe detecting dyes when dissolved in different solvents.

Example 28 Testing of Indicator with Viruses

Virus testing was carried out by Gibraltar Laboratories (Fairfield,N.J.) under the direction and supervision of one of the inventors. Poliovirus type 1 and Rhinovirus were prepared and inoculated into MA-104embryonic monkey kidney cells propagated and fed with Dulbecco'sModified Eagle's Medium (DMEM), supplemented with fetal calf serum to aconcentration of 5% and Incubated at 37° C.±1° C. in its presence of 5%CO₂ for 6 days. Viral propagation was detected by microscopicobservation of infected cell sheets for cellular disintegration(cytopathic effect, CPE) such as rounding, crenation, lysis, pyknosis,etc. as observed in at least 50% of the cell sheet. Cytotoxicity wasmeasured as the extent of cellular disintegration as produced by theagent alone without the virus. Virus was titrated using ten-fold serialdilutions in DMEM, 4 replicates MA 104 cultures per dilution, eachreplicate inoculated with 0.1 of virus dilution. The extent of viralreplication was calculated as the tissue culture infectious dose-50%(TCID 50) as determined by the method of Reed and Muench.

Reichardt's dye-coated stickers (160 mg/10 mL acetonitrile, 80 mg/10 mLacetonitrile, 40 mg/10 mL acetonitrile, and 20 mg/10 mL acetonitirile)were used as a test surface. 50 μl of undiluted virus (10⁻¹⁰/50 μl forPolio virus and 10⁻⁹/50 μl for Rhinovirus) in media was dropped ontoeach sticker and allowed to stand for five minutes before removing thedroplet with a cotton swab. Aliquots of media alone, virus-free cellculture media, and virus-free cell culture saline were utilized ascontrol samples and also allowed to stand for five minutes beforeswabbing. For poliovirus, the saline control appeared to interfere withtechnology, while the media did not cause de-colorization. Dilutions ofPolio virus in media were therefore used for the remainder of theexperiment. The virus was diluted serially in media in ten-foldincrements and 50 μl aliquots applied to each sticker. After allowing tostand for five minutes, the droplets were swabbed off the sticker. ForRhinovirus, the media control was found to interfere while the salinecontrol did not lead to de-colorization of the dye. Thus, ten-foldserial dilutions of the virus diluted in saline were applied to thestickers in 50 μl aliquots and swabbed after five minutes. For bothPolio virus and Rhinovirus, the stickers were de-colorized down to 10⁻⁶(i.e. the sixth in the series of ten-fold dilutions), indicating thatthe dye-coated stickers possess sensitivity towards the detection ofthese viruses.

Two solutions of Reichardt's dye (80 mg/10 mL acetonitrile with orwithout 400 μl TWEEN 80 surfactant) were prepared. A 100 μl drop ofeither Polio virus or Rhinovirus (both undiluted in media) was pipettedonto a folded Scott towel and drops of the Reichardt's dye were added toeach of the virus-containing spots. The color was rapidly discharged forboth surfactant- and non-surfactant-containing solutions. Dye waseventually added until the color persisted (approximately 9 drops). Thesame media and saline controls mentioned previously were also tested.Though media did exhibit some ability to de-colorize the dye, the salinepresented the same titration behavior previously observed with water.

COMPARATIVE EXAMPLES Not Examples of the Invention

Aged chicked was used as a bacteria source for comparative examples. Thetransparent film covering half of a fresh chicken on a polystyrene tray(from supermarket) was stored at room temperature for three weeks. Thepale yellow juices that collected in the polystyrene tray were collectedusing a pipette and used for tests.

Comparative Example 1

Aged chicken juice was dropped onto a SCOTT® paper towel. CI Acid Green41 (from Aldrich Chemical) solution (0.008 mol/l) (an example of anhydroxyanthraquinone dye) structure 27 below was dropped onto the agedchicken juice. No color changes were observed. As a control, 100 mg ofReichardt's dye was suspended in 10 ml acetonitrile. This suspension wasdropped onto the aged chick juice and immediately decolorized.

Comparative Example 2

Aged chicken juice was dropped onto a SCOTT® paper towel. Cl Acid Green25 solution (0.008 mol/l), structure 28 below, an example of ananthraquinone dye, was dropped onto the aged chicken juice. No colorchanges were observed. As a control, 100 mg of Reichardt's dye wassuspended in 10 ml acetonitrile. This suspension was dropped onto theaged chick juice and immediately decolorized.

Comparative Example 3

Aged chicken juice was dropped onto a SCOTT® paper towel. 50 mg of CIAcid Red 37 (from Aldrich Chemical), structure 29 below and example ofan aminoazo dye, was dissolved in 10 ml deionized water. This dyesolution was dropped onto the aged chicken juice on the paper towel. Nocolor changes were observed. As a control, 100 mg of Reichardt's dye wassuspended in 10 ml acetonitrile. This suspension was dropped onto theaged chick juice and immediately decolorized,

Comparative Example 4

Aged chicken juice was dropped onto a SCOTT® paper towel. 50 mg of CIAcid Yellow 23 (also known as the food colorant tartrazine) (fromAldrich Chemical), structure 30 below, and example of a phenylpyrazolonedye, was dissolved in 10 ml deionized water. This dye solution wasdropped onto the aged chicken juice on the paper towel. No color changeswere observed. As a control, 100 mg of Reichardt's dye was suspended in10 ml acetonitrile, This suspension was dropped onto the aged chickjuice and immediately decolorized.

Comparative Example 5

Aged chicken juice was dropped onto a SCOTT® paper towel. CI Acid Red 52(sulforhodamine B), structure 31 below, an example of a xanthene dye,solution in water was dropped onto the aged chicken juice on the papertowel. No color changes were observed. As a control, 100 mg ofReichardt's dye was suspended in 10 ml acetonitrile. This suspension wasdropped onto the aged chick juice and immediately deodorized.

Comparative Example 6

Aged chicken juice was dropped onto a SCOTT® paper towel. 30 mg of CIAcid Blue 74 (also known as Indigo Carmine), structure 32 below, (fromAldrich Chemical), as an example of an indigoid dye, was dissolved in 10ml deionized water. This dye solution was dropped onto the aged chickenjuice on the paper towel. No color changes were observed. As a control,100 mg of Reichardt's dye was suspended in 10 ml acetonitrile. Thissuspension was dropped onto the aged chick juice and immediatelydecolorized.

As will be appreciated by those skilled in the art, changes andvariations to the invention are considered to be within the ability ofthose skilled in the art. Examples of such changes are contained in thepatents identified above, each of which is incorporated herein byreference in its entirety to the extent it is consistent with thisspecification. Such changes and variations are intended by the inventorsto be within the scope of the invention. It is also to be understoodthat the scope of the present invention is not to be interpreted aslimited to the specific embodiments disclosed herein, but only inaccordance with the appended claims when read in light of the foregoingdisclosure.

1-20. (canceled)
 21. A method for detecting bacteria on a surface, themethod comprising contacting the surface with a substrate coated with acomposition comprising a zwitterionic microbe-sensitive colorant; anddetecting a visible color change that indicates the presence ofbacteria.
 22. The method of claim 21, wherein the composition comprisesa mobile phase.
 23. The method of claim 22, wherein the mobile phaseincludes a liquid or gel.
 24. The method of claim 21, wherein thecolorant is Reichardt's dye.
 25. The method of claim 21, wherein thecolorant has the general structure:

wherein, n is 0 or greater; and X is oxygen, carbon, nitrogen,phosphorous or sulfur.
 26. The method of claim 21, wherein the coloranthas the general structure:

wherein, n is 0 or greater; and R is alkyl, aryl, or phenyl.
 27. Themethod of claim 21, wherein the colorant is selected one of thefollowing structures:


28. The method of claim 21, wherein the composition is present in anamount from about 0.01 to about 10 weight percent on a dry basis. 29.The method of claim 21, wherein the composition is present in an amountof from about 0.05 to about 5 weight percent on a dry basis.
 30. Themethod of claim 21, wherein the substrate is a film.
 31. The method ofclaim 21, wherein the substrate contains fibers.
 32. The method of claim31, wherein the fibers include meltblown fibers, spunbonded fibers, or acombination thereof.
 33. The method of claim 31, wherein the fibersinclude cellulosic fibers.
 34. The method of claim 21, wherein the colorchange occurs in less than one minute after exposure to the bacteria.35. The method of claim 21, wherein the color change occurs at a rateproportional to the concentration of the bacteria.
 36. The method ofclaim 21, wherein the bacteria are gram negative bacteria.
 37. Themethod of claim 21, wherein the bacteria are gram positive bacteria.